Smooth muscle cells (SMCs), often termed the most primitive type of muscle cell because they most resemble non-muscle cells, are called “smooth” because they contain no striations, unlike skeletal and cardiac muscle cells. Smooth muscle cells aggregate to form smooth muscle which constitutes the contractile portion of the stomach, intestine and uterus, the walls of arteries, the ducts of secretory glands and many other regions in which slow and sustained contractions are needed.
Abnormal gene expression in SMC plays a major role in numerous diseases including, but not limited to, atherosclerosis, hypertension, stroke, asthma and multiple gastrointestinal, urogenital and reproductive disorders. These diseases are the leading causes of morbidity and mortality in Western Societies, and account for billions of dollars in health care costs in the United States alone each year.
In recent years, the understanding of muscle differentiation has been enhanced greatly with the identification of several key cis-elements and trans-factors that regulate expression of muscle-specific genes. Firulli A. B., et al., 1997, Trends in Genetics, 13:364-369; Sartorelli V. et al., 1993, Circ. Res., 72:925-931. However, the elucidation of transcriptional pathways that govern muscle differentiation has been restricted primarily to skeletal and cardiac muscle. Currently, no transcription factors have yet been identified that direct smooth muscle-specific gene expression, or SMC myogenesis. Owens G. K., 1995, Physiol. Rev., 75:487-517. Unlike skeletal and cardiac myocytes, SMC do not undergo terminal differentiation. Furthermore, they exhibit a high degree of phenotype plasticity, both in culture and in vivo. Owens G. K., 1995, Physiol. Rev., 75:487-517; Schwartz, S. M. et al., 1990, Physiol. Rev., 70:1177-1209. Phenotype plasticity is particularly striking when SMC located in the media of normal vessels are compared to SMC located in intimal lesions resulting from vascular injury or atherosclerotic disease. Schwartz, S. M. et al., 1990, Physiol. Rev., 70:1177-1209; Ross R., 1993, Nature, 362:801-809; Kocher O. et al., 1991, Lab. Invest., 65:459-470; Kocher O. et al., 1986, Hum. Pathol., 17:875-880. Major modifications include decreased expression of smooth muscle isoforms of contractile proteins, altered growth regulatory properties, increased matrix production, abnormal lipid metabolism and decreased contractility. Owens G. K., 1995, Physiol. Rev., 75:487-517. The process by which SMC undergo such changes is referred to as “phenotypic modulation”. Chamley-Campbell J. H. et al., 1981, Atherosclerosis, 40:347-357. Importantly, these alterations in expression patterns of SMC protein cannot simply be viewed as a consequence of vascular disease, but rather are likely to contribute to the progression of the disease.
Expression of smooth muscle myosin heavy chain (SM-MHC) appears to be completely restricted to SMC lineages throughout development (Miano J. et al., 1994, Circ. Res., 75:803-812). To date, four SM-MHC isoforms (SMC-1A, SMC-1B, SMC-2A, and SMC-2B) have been identified (Nagai R. et al., 1989, J. Biol. Chem., 264:9734-9737; White S. et al., 1993, Am. J. Physiol., 264:C1252-C1258; Kelley C. A. et al., 1993, J. Biol. Chem., 268:12848-12854), all of which are derived from alternative splicing of a single gene (Miano J. et al., 1994. Circ. Res., 75:803-812; Babij P. et al., 1989, J. Mol. Biol., 210:673-679). Alterations in expression of SM-MHC isoforms have been extensively documented in SMC that have undergone phenotypic modulation either when placed in culture (Rovner A. S., 1986, J. Biol. Chem., 261:14740-14745; Kawamoto S. et al., 1987, J. Biol. Chem., 262:7282-7288), or in vascular lesions of both humans and several animal models of vascular disease (Aikawa M. et al., 1997, Circulation, 96:82-90; Sartore S, et al., 1994, J. Vasc. Res., 31:61-81).
Transcriptional regulation of the SM-MHC gene has been analyzed in cultured SMC and several functional cis-elements have been identified. White S. L. et al., 1996, J. Biol. Chem., 271:15008-15017; Katoh Y. et al., 1994, J. Biol. Chem., 269:30538-30545; Watanabe M. et al., 1996, Circ. Res., 78:978-989; Kallmeier R. C. et al., 1995, J. Biol. Chem., 270:30949-30957; Madsen C. S. et al., 1997, J. Biol. Chem., 272:6332-6340; Madsen C. S. et al., 1997, J. Biol Chem., 272:29842-29851. However, because differentiation of SMC is known to be dependent on many local environmental cues that cannot be completely reproduced in vitro, cultured SMC are known to be phenotypically modified as compared to their in vivo counterparts (Owens G. K., 1995, Physiol. Rev., 75:487-517; Chamley-Campbell J. H. et al., 1981, Atherosclerosis, 40:347-357). As such, certain limitations may apply regarding the usefulness of cultured SMC in defining transcriptional programs that occur during normal SMC differentiation and maturation within the animal.
A few promoters relating to smooth muscles have been described in the art, e.g., promoters for SM-actin and SM22 genes. However, a major disadvantage with these promoters is that they are clearly not SMC specific. SM22 and SM-actin are highly expressed in myofibroblasts during wound repair, within granulomatous tissues, tumors, etc. The promoters for these genes are also transiently activated in skeletal and cardiac muscle during development, and in association with a number of pathological circumstances (e.g. myocardial hypertrophy). In addition, the SM22 promoter fragments tested to date also have very little activity in SMC tissues of adult mice. Thus, such promoters have major limitations in terms of their utility in smooth muscle tissue specific targeting and expression in vivo.
Thus, there is a need in the art for transcription regulatory sequences (e.g., promoters and enhancers) that can direct gene expression specifically in smooth muscle tissues in vivo (e.g., in human or non-human animals). There is also a need for relatively small smooth muscle specific promoter/enhancers that retain high level SMC specific expression in vivo and yet are selectively active in subsets of SMC (e.g. vascular versus gastrointestinal SMC, large versus small arteries, pulmonary versus gastrointestinal SMC, etc.). Methods for utilizing such SMC specific promoters and enhancers to target delivery and expression of polynucleotide to SMCs are also needed. The present invention fulfills these and other needs.